..
`.
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intcmatio'iiill Bureau
`' . ;
`I
`
`PCT
`INTERNATIONAL APPLICATION PUBLISHED. UNDER urn PATENT COOPERATION TREATY (PCJ)
`WO 91/11457
`(51) International Patent Classification 5 :
`_{it) Intematiqnal Publication Number: ·
`C07K 7 /34, 7 /10, A61K 37 /OZ
`A61K37/28
`
`Al
`
`(43) Intemationai ~blication Date:
`
`8 August 1991 (08.08.91)
`
`(21) International Application Number:
`
`(22) International Filing Date :
`
`PCT/US91/00500
`
`(74) Agents: MURASHIGE, Kate, H. et al.; Irell & Ma~ella,
`S45 Middlefield Road, Suite 200, Menlo Park, CA 94025
`24 January 1991 (24.01.91) ·
`(US).
`·
`
`(30) Priority data:
`468,136
`
`24 January 1990 (24.01.90)
`
`US
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`us
`Filed on
`
`468,736 (CIP)
`24 January 1990 (24.01.90)
`
`(71)(72) Applicants and Inventors: BUCKLEY, Douglas, I. [US/
`US]; 215 Brookwood Road, Woodside, CA 94062 (US).
`HABENER, Joel, F. [US/US]; 217 Plymouth Road,
`Newton Highlands, MA 02161 (US). MALLORY,
`Joanne, B. [US/US]; 243 Acalanes, Apt. 9, Sunnyvale,
`CA 94086 (US). MOJSOV, Svetlana [YU/YU]; 504 East
`63rd Street, New York, NY 10021 (US).
`.
`
`(81) Designated States: AT (European patent), BE (European
`· patent), CA, CH (European patent), DE (European pa(cid:173)
`tent), DK (European patent), ES (European patent), FR
`(-European patent), GB (European patent), GR (Euro(cid:173)
`pean patent), IT (European patent),. JP, LU (European
`patent), NL (European patent), SE (European patent),
`us.
`
`Published
`With international search report.
`
`(54) Title : GLP-1 ANALOGS USEFUL FOR DIABETES TREATMENT
`
`(S7) Abstract
`
`The invention provides effective analogs of the active GLP-1 peptides, 7-34, 7-35, 7-36, and 7-37, which have improved
`characteristics for treatment of diabetes Type II. These analogs have amino acid substitutions at positions 7-10 and/or are tnm(cid:173)
`cated at the C-tenninus and/ or contain various other amino ~cid substitutions in the basic peptide. The analogs may either have
`an enhanced capacity to stimulate insulin production as cottfpared to gluc'agon or may exhibit enhanced stability in plasma as
`compared to GLP-1 (7-37) or both. Either of these properties will enhance the potency of the analog as a therapeutic. Analogs
`having D-amino acid substitutions in the 7 and 8 positions and/or N-alkylated· or N-acylated amino acids in the 7 position are
`·
`particularly resistant to degradation in vivo.
`
`FRESENIUS EXHIBIT 1041
`Page 1 of 50
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`

`

`i
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`"
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`Ausuia
`AU
`AliSlralia
`BB
`Barbados
`BE
`Belgium
`BF
`Burkina Faso
`BG
`Bulgaria
`BJ
`Benin
`BR
`Brazll
`CA
`Canada
`CF
`Central African Republic
`CG . Congo
`CH
`Switzerland
`C6te d 'lvoirc
`Cl
`CM
`Cameroon
`cs
`Cze«;hoslovakia
`DE
`Germany
`DK
`Denmark
`
`ES
`Fl
`FR
`GA
`. CB
`CN
`CR
`HU
`IT
`. JP
`KP
`
`Spain
`Finland
`Franw
`Oabon
`United Kingdom
`Ouinca
`Oreece
`Hungary
`Italy
`Japan
`Democratic People's Republic
`of Korc:.i
`KR
`Republic-or Korea
`LI
`Uc:ch1en,1t:in
`LK
`Sri Lanka
`LU
`Luxembourg .
`MC Monaco
`
`MG
`Madagascar
`Mali
`ML
`MN
`Mongolia
`MR
`Mauritania
`MW Malawi
`NL
`Netherlands
`NO
`Norway
`PL
`Poland
`RO
`-Romania
`SD
`Sudan
`SE
`Sweden
`SN
`Slmegal
`SU
`Soviet Union
`TD
`Chad
`TC
`Togo
`us
`United Staws of America
`
`FRESENIUS EXHIBIT 1041
`Page 2 of 50
`
`

`

`W091/11457
`
`PCT/US91/00S00
`
`-1-
`
`5
`
`GLP-1 ANALOGS USEFUL FOR DIABETES TREATMENT
`'·-
`This is· a contt.i1.1uation-'in-part of U.S.
`Application Serial No. 4·68, 736, filed 24 January 1990.
`
`10 Technical Field
`The .invention relates to the f i eld of improved
`pha.rmaceutical co.mpositi ofis. Specifically, the invention
`concerns analogs of the g.lucagon-like peptide I fragment
`7-36 or 7-37 with impro~d pharmacological properties.
`
`15
`
`20
`
`' .
`
`.!
`
`~
`
`·•:·
`
`+
`
`•
`
`;
`
`'
`
`" •!I.,
`
`Background Art
`Glucose metabo1ism is regu.iated by a number of
`peptide hormones, includihg iris~in~ glucagon, and
`gastric inhibitory ·'peptide . (GIP). · Tl1e complex mechanism
`by which these peptld~ ' h~rmones regulate this metabolism
`·and the manner in which tihey affe·c::~:: each other is at
`.
`.
`.
`least partially elucidated. For.}e-xample, glucagon binds
`to receptors on the surface of the pancreatic beta cells
`which produce insul:in, and stimu.1.ates in5ulin secretion.
`25 Glucagon-like peptide I" has been suggested to stimulate
`.
`. 1
`insulin secretion but· ~his has not:"been confirmed.
`several of the~--hormonas·t·ori•~:inate from a mam(cid:173)
`malian glucagon pre·cursor "prog:l..'?"cagoh'" · which is a 180
`amino acid peptide:· · Prot~olysis, a~d process1ng of this
`peptide results i .n a number of the~ prot~ih hormones;
`the results of the processing dep~nd on the origin of the
`.
`.
`cells in which .this•. 0¢c1.Ws ~ For eximple, in ~he pig and
`.r ·at pancreas, pr.og1u·cagor( is -processed to form glucagon
`and glicentin-related pan9reatie ··peptide, a large peptide
`
`•
`
`30
`
`..
`
`~
`
`35
`
`.
`. ~ .... ~
`, i. • • " ff
`
`..;
`
`.r...
`
`FRESENIUS EXHIBIT 1041
`Page 3 of 50
`
`

`

`W091/11457
`
`PCT lUS91/00500
`
`-2-
`
`: in.
`which contain·s both GLP-1 and GLP-2 s·e9uences :
`porcine small intestine, the secreted products are the 69
`amino acid glucagon-containing peptide glicentin and the
`two glucagon-like sequences,GLP-1 and GLP-2 as separate
`peptides.
`
`5
`
`~
`
`10
`
`15
`
`In any event, however, the overall sequence of
`proglucagon contains the 29·amino acid sequence of
`glucagon,. the 36 or 37 amino acid sequence of GLP-1 and
`the 34 amino acid sequence of GLP-2; separated by amino
`acid spacer sequences.
`Early attempts to assign a pattern of activity
`to GLP-1 gave ambiguous results, and it was subsequently
`concluded that truncated forms of this peptide. are bio(cid:173)
`logically active. Mojsov, S., et al. J Clin Invest
`(1987} 79:616-619 disclose that only the 31 amino acid
`peptide GLP-1 (7-37) strongly stimulates the release of
`insulin from pancreas; although both the truncated and
`full length 37 amino acid form had earlier been found in
`pancreas and intestine. It has been demonstrated that
`20 GLP-1 (7-36}, possibly with the carboxy terminus
`amidated, is also a potent mediator of insulin release.
`(See, e.g., Holst, J.J., et al. FEBS Letters · (1987)
`211:169-174).
`The invention described below concerns analogs
`of these truncated· forms of GLP-1, which have desirable
`combinations of characteristics as they relate to potency
`in potentiating glucose-induced insulin secretion and
`glucose-induced inhibition of glucagon secretion and to
`circulating half-life. The physiological effects of the
`truncated forms in potentiating glucose-induced insulin
`secretion have been shown as described above by Holst,
`J .J., et al. and Mojsov, s., et . al. (supra). · The
`. activity of the truncated hormones in iphibiting glucagon
`release has been shown by orskov, c., et al. Endocrinol
`
`25
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`30
`
`35
`
`FRESENIUS EXHIBIT 1041
`Page 4 of 50
`
`

`

`WO 91/11457
`
`PCT/US91/00500
`
`-3-
`
`f
`
`· . a
`
`.
`
`.
`
`i , ,· . • ,. t .
`
`. \ ,
`
`. · ·""" '
`
`·.·
`
`,
`
`. '
`
`, .
`
`•
`
`•
`
`I.
`
`••
`
`:
`
`•'
`
`•
`
`· ~ ......
`
`':
`
`.
`
`.
`
`(1°988) ill,:2009-201_3; Suzuki, s., . eF al. Diabetes
`. . ;.,.
`.
`Research; Clinical Practice (1988} a(Supp. 1}:S30. The
`circulating half-life of these ~runcated forms is
`short--approxiinately four m±nute$ as shown by Kreymann et
`.
`al. The Lancet (December .~~ 1987) ~300-1303. The
`modified forms of these 1rruncated GLP-1 peptides provide
`the opportunity to optiaj..ze ·these properties.
`Ther~ is som~ .. lriteratu~~ r~lating to the study
`of degradation ~f peptidff:.~pnnones ·J~ the liver and in
`plasma and the half-life .of such ,,~ormones in vivo
`. .
`.
`: .
`'l,,, . ': ' :'·
`generally. An early paper ,by McD'otia.ld, J .1{. et al., J
`.. ~
`. . ·
`Biol Chem (1969) 2.il:619~,"."6208 ~aowed t~at a dipeptidase
`..
`.. .
`... ;. , • . .
`.
`was responsible fo~ th~ degradatton 'of glucagon in rat
`.
`· . .. •:
`... -.~
`:.:· ·
`liver. Studies on t~e gi'.Qwth hormone rel,easing factor, a
`15 member of the general glµ.cagon, .GI,.B-1, GLP-2 family, was
`. ' ;
`. '.
`. .
`. ~
`.
`shown to be rapidly degr.aded in .. plasma ln vitro and also
`... . ,
`in vivo by a dipeptidase, (F:ronman, L.A. et al., J Clin
`Inyest (1986} 78:9.06~913) .• ·)!urphy, · W.A. et al., in
`Peptide Research (i!l88} ,_,l·~ 36~41; '" showed that some but not
`all alkylated grQwth horin:one releas~ng factor peptides
`had higher potency in vf-cto· •· In particular, for example,
`,. •
`,r~. •
`.
`the triisopropylated GRF~'29 was found to be 106 times
`more active than q!U'-29 i~self. On ~he other hand, GRF- ·
`29 which.was in methylated at the N-terminus was only 40%
`as potent as the parent.
`· It was also shown that
`substitution of D-Ala __ position 2 of this hormone enhanced
`its potency. It was, of course, .not certa~n to what
`effect on properties t~~ enhance~ent of potency could be
`attributed.
`Others have at~empted so•e modifications of
`GLP-1 (7-37). It h~s bee!l s.hown that deletion of the
`histidine residue at position 7 greatly diminishes the
`activity of th~ hormon~ .. (Suzuki, s., et al,. (supra);
`' .... .
`-
`Hendrick, G.K.; et al. Abstract: Ensocrine Society
`
`t
`
`•
`
`•
`
`..
`
`. ,
`
`•
`
`.
`
`.
`
`"I· .
`
`:
`
`5
`
`10
`
`20
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`30
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`
`FRESENIUS EXHIBIT 1041
`Page 5 of 50
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`

`

`W091/11457
`
`PCT /US91/00500
`
`-4-
`
`Meeting, New Orleans, LA (1988)). There have been
`conflicting reports concerning the effect of one or more
`c-terminal deletions (Suzuki, s., et al. (supra);
`Yanaihara, c., et al. Abstra;c:t for A Glucagon and Related
`Peptides Satellite Symposium, 8th International congress
`of Endocrinology, July 15-16, 1988, Osaka, Japan).
`However, there is an extensive literature with regard to
`-
`modifications of other members of this peptide hormone
`family, such as GIP, glucagon releasing factor (GRF),
`secretin and vasoactive intestinal peptide (VIP).
`
`Disclosure of the Invention
`The invention provides modified forms of the
`GLP-1 (7-34); (7-35); (7-36) or (7-37) human peptide or
`the c~terminal amidated forms thereof. The native
`peptides have the amino acid sequence:
`
`25
`20
`15
`10
`7
`H-A-E-G-T-F-T-s-o-v-s-s-Y-L-E-G-Q-A-A-
`
`37
`30
`K-E-F-I-A-W-L-V-K-(G)-(R)-(G)
`
`wherein (G), (R), and (G) are present or absent depending
`on indicated chain length. The modified forms contain
`one or more alterations of the native structure and are
`of improved ability for therapeutic use. Either the
`modified forms have greater potency than glucagon to
`potentiate insulin secretion or enhanced stability in
`plasma or both. This potency and enhanced sta.bility can
`be assessed as described below.
`The standard one lett~ ~bbreviation code for amino
`acids is used.
`The analogs of the invention which -show enhanced
`insulin stimulating properties have the foregoing
`
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`

`

`W091/11457
`
`PCT/US91/00500
`
`-s-
`
`at least one of:
`
`sequence, or the c-terminal amide thereof, with at least
`one modification selected from the group consisting of:
`(a) substitution of· a neutral amino acid, arginine,
`.
`.
`or a D form of lysine for lysin~ ·at position 26 and/or 34
`and/or a neutral amino ·acid, lysine, or a D form of
`arginine for arginine at j>osition 36;
`(b) substitution of an oxidation-resistant amino
`.
`'
`acid for tryptophari at positfon 31;
`substitution according to
`(c)
`y for Vat position 16;
`K for s at position 18;
`D for E at position 21;
`s for G at posi~ion 22;
`R for Q ·at position 23;
`R for A at position 24; and
`Q for Kat position 26;
`(d) a substitution __ comprising at least one of:
`an alternative"·¢~all neutral amino acid for A
`at position 8; .
`an alternative acidic a~ino acid or neutral
`amino acid for E at .. position 9;
`an alternative neutral amino acid for G at
`position 10; and
`an alternative ·acidic amino acid for D at
`positio~ 15; and ·
`(e) substitution of an alternative neutral amino
`acid or the Dor N-acylated or alkylated form of
`histidine for histidine at position 7.
`(b), (d) and
`With respect to · mo.di~ications (a),
`(e), the substituted ·amino acids t~ay be
`in the D form, as
`.
`. ·
`t
`indicated by a super~cript t, e.g., C.
`1he amino acids
`substituted at position 7 can also be in the N-acylated·
`or N-alkylated. forms.
`
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`

`

`W091/11457
`
`PCT /US91/00500
`
`-6-
`
`5
`
`10
`
`Thus, · one aspect of the invention is directed ·to
`peptides having enhanced insulin stimulating properties
`analogous to the above-mentioned truncated forms of GLP-1
`(7-34) to GLP-1 (7-37), as ~escribed above.
`In another aspect, the invention is directed to
`peptides which show enhanced degradation resistance in
`plasma as comparedto GLP-1 (7-37) wherein this enhanced
`resistance to degradation is defined as set forth below.
`In these analogs, any of the above-mentioned truncated
`forms of GLP-1 (7-34) to GLP-1 (7-37) or their c-terminal
`amidated forms is modified by
`(a) substitution of a D-neutral or D-acidic amino
`acid for Hat position 7, or
`(b) substitution of a D-amino acid for A at
`position 8, or
`(c) both, or
`(d) substitution of an N-acylated or N-alkylated
`form of any naturally occurring amino acid for Hat
`position 7.
`Thus, analogs of the invention which are resistant
`to degradation include (N-acyl (l-6C) AA) 7 GLP-1 (7-37)
`and (N-alkyl (1-6C) AA) 7 GLP-1 (7-37) wherein when AA i s
`a lysyl residue, one or both nitrogens may be alkylated
`or acylated. AA symbolizes any amino acid consistent
`25 with retention of insulin stimulating activity.
`For substitutions of D-amino acids in the 7 and 8
`positions, the D residue of any acidic or neutral amino
`acid can be used at position 7 and of any amino acid at
`position 8, again consistent with insulin stimulating
`activity. Either or both of position 7 and 8 can be
`substituted by a D-amino acid; .the D-amino acid at
`position 7 can also be acylated or !ilkyl_ated as set forth
`above. These modified forms are applicable not only to
`
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`FRESENIUS EXHIBIT 1041
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`

`

`WO91/11457
`
`PCT/US91/00500
`
`-7-
`
`.
`.
`.
`..
`GLP-1 (7-37) but also the·shorter truncated analogs as ·
`set forth above.
`In other aspects, the invention is directed to
`pharmaceutical compositions--containing one or more of
`... ,
`these peptides as active .!:.ingredients and to methods to
`treat Type II diabetes using these 'peptides or
`compositions thereof.
`
`Brief Description of the Drawings ~.
`Figure 1 schematic~lly outlines the classification
`of amino acids as used herein.
`· · :-:·
`Figure 2 gives a-·,-1ist of various compounds of the
`invention.
`Figure 3 sho~ the · results iof '. radiolabel sequencing
`analysis for degradatio.ti; df two · ~alogs in plasma.
`Figure 4 shows tl:ieresults ; of various GLP-1 (7-37)
`, .
`analogs with changes in the amino. terminal region, to
`displace 1251-GLP-1 (7-39)'-from ali'ti:no terminal specific
`antiserum.
`
`Modes of Carrying out the .Invention
`The analogs of- the invention, which are modified
`forms of the · GLP-1·(7-34), · (7~35), (7-3,6) or (7-37) are
`characterized by showingd:g-re-ater po.tency · than · glucagon in
`an in vitro assay measu:rlig insuli,n release from isolated
`rat .islets in culture, or ·by enhanced stability in plasma
`or both.
`Assays for Anal.oqs with Enhanced Insulin Release
`stimulating Properties · ; .. ·.: ·
`,
`'
`l:
`One group of analogs .of the invention is more
`potent than glucagon in ·stimulating,insulin release from
`islet cells. - By being "m:ore po"t"ent than glucagon in
`stimulating insulin rel~ from isl.et cells" is meant
`that the analog referred .to shows greater potenc:y in an
`
`,,
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`

`W091/11457
`
`PCT /US91/00500
`
`-s-
`
`in vitro assay selected from the group consisting of the
`following: Rat islets for these assays are isolated by
`the method of Sutton, R. et al., Transplantation (1986)
`.!a,:689-691, incorporated herein by reference. Briefly,
`Sprague-Dawley male rats are anesthetized and the lower
`end of the common bile duct is cannulated with a 2 FG
`cannula tied in place. The·left and right hepatic ducts
`-
`are then ligated separately above the region of the entry
`of pancreatic ducts into the biliary tree. · The rats are
`killed by exsanguination and 3 mL Hank's solution
`containing 7.5 mM cac12, 20 mM HEPES buffer and 1-6 mg/mL
`Type I collagenase are run into the cannula to uniformly
`distend the pancreas. The pancreas is then excised and
`placed in a beaker on ice prior to incubation in Hank's
`solution containing 20 mM HEPES buffer at 37°c.
`After 13-25 min of incubation, the pancreas is
`removed and placed _in Hank's solution containing s g/1
`bovine serum albumin and 20 mM HEPES buffer at 4°c. All
`of the pancreatic tissue is then gently syringed through
`a 14 FG needle, suspended in further Hank's solution
`containing HEPES as above, centrifuged at 50 g for 10 sec
`and the supernatant is discarded. The tissue pellet is
`resuspended and again gently syringed, · followed by
`another wash, after which the dispersed tissue is passed
`through a nylon mesh filter of 500 u pore size. The
`filtered tissue is centrifuged at 350 g for 5 sec, the
`supernatant discarded, and the tissue is then suspended
`in 25% Ficoll made up in Hank's with H:EPES as above, on
`which was layered a discontinuous density gradient of
`23%, 20%, and 11% Ficoll solutions. This density
`gradient was spun at 750 g for 10 min at 4°c, and the .
`t _issue obtained from -the upper _two interfaces ·was washed
`tn.ree times in Hank I s s.olution .arid vieweq through · a
`dissecting microscope for hand picking of· islets.
`
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`

`WO91/114S7
`
`PCT/US91/00SO0
`
`-9-
`
`5
`
`In one · approach :the-.ability _of the GLP,.;,1 analog to
`potentiate secretion from _these Is~ets is then determined
`according to the_ method <>:-f ~chatz, H. et al., in "Methods
`in Diabetes Researcn~• , (.~~:4 ,.- Volume ) ,., Part c: pages
`291-307, incorpora~edh,er,in by -reference.
`In this
`method, 5-10 islets per ;est tube are incubated in l mL
`Krebs-Ringer-bicarbpnate ~uffer (KRBbuffer). For
`testing, glucagon or the modified an~log of the invention
`is added at 5-10 µg/~. .· The level of insulin released
`1 O may be measured by _ _'the .. method of Jensen, S. L. et al. , M J
`Physiol (1978) -235:E381-~8-6, incorporated. herein by
`reference.
`
`~ .
`
`:I
`
`I •
`
`•
`
`•
`
`During the 60 min preincu~ation, the experiment is
`set up, so that all that is needed at the end of the
`
`35
`
`The following, protocol is a preferred method to
`15 measure stimulation or insulin ·secretion. After
`collagenase digestiol), .the isl~~s are allowed to recover
`overnight by incubatj,on in-DMEM (Dulbecco•s Modified
`Eagle Medium 16 w/o glucose), 2.8 mM glucose, 10% fetal
`bovine serum (FBS): at 31;9-c,_· 5% CQ2'.
`The next .day, isiets to -be used for the experiment
`are transferred t9 DMEM, 'hQ glucot;;-e, o .. 2% BSA (Armour,
`clinical grade, maq.e at 5~ stock) -tor a 60 min
`Islets
`preincubation in serum~tree, glucose-free medium.
`are picked up by Eppemio~ pipette ·and ,transferred to 60
`mm TC plates containing s:,. o mL medium and returned to the
`incubator for 60 min._ Islets are counted duri,ng this
`transfer.
`(Note: each d~ta pei~t is 5 islets,
`experiments are usually -performed in quadruplicate;
`therefore, 20 islets are . u_~ed per ~ata point.)
`Typically, recoveries -ar~ 150-2QO :i..slets per pancreas.
`Any suspect islets--too r .agged or falling apart--are not
`used.
`
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`

`

`~
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`.-,.
`
`WO91/11457
`
`PCT/US9l/00500
`
`-10-
`
`preincubation is to transfer islets .1.n groups of 5 to
`experimental conditions. The experiment is set up in 48
`well TC plates with O. 5 mL medium per well. To OMEM-0. 2 %
`BSA is added glucose to desired concentration (usually
`2.8 mM for hypoglycemic conditions, 5.6 mM glucose for
`euglycemic, or 16.7 mM glucose for hyperglycemic) and
`test compound at various dose ranges (typically, 1 pM to
`100 nM). Test compound is diluted from stock stored at
`-so0 c and at -0.3 mM serially into phosphate buffered
`saline (PBS) 0.2% BSA to prevent loss on.· sides of tubes.
`After medium plus test compound is mixed, -o.5 mL each is
`added to 4 wells for quadruplicate data points.
`After the preincubation period, 5 islets are added
`per well.
`Islets are picked up by eppendorf pipette in
`25 ul volume.
`Incubation continues another 60 min, at
`which time 0.3 mL is harvested per well with care taken
`not to pick up islets. Wells are then rechecked for
`islet number. Medium is then assayed for insulin content
`using an insulin RIA.
`If medium is not immediately
`assayed, it is stored at -20°c until assay. Dose
`response curves for insulin secretion are plotted and
`Eo50 is calculated from the curves.
`Higher potency as compared to glucagon is defined
`as either higher levels of insulin released by the analog
`using the same concentrations of glucagon and analog or,
`alternatively, the same level of insulin release but
`using a lower concentration of analog than glucagon.
`While the foregoing assays form specific criteria
`for judging enhanced potency, alternative assays can also
`be used as substitutes for those set forth above.
`An additi~nal test for potency of the compounds of
`the i~vention measures thei~ ability to -stimulate cAMP
`product_ion in RIN 1046-38 cells • . This assay can be
`conducted as follows:
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`FRESENIUS EXHIBIT 1041
`Page 12 of 50
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`

`WO 91/11457
`
`PCT /US91/00500
`
`. .. -11-
`
`-..:,.
`
`~,l
`
`.
`
`I
`~
`
`J
`
`.
`
`•
`
`:
`
`On day 1 , 5 x 105 R'.ru 1·046-38 c::ells (Drucker, D.J.,
`et al., Proc Natl .Acad sdi USA (1987} 84:3434-3438) are
`seeded into indi~f~u~l w~lls of 6-~~ll dishes with 2.5 mL
`Ml99 culture medium. Oh 'day- 4, ~ells are re-fed with
`fresh medium and on ·day' s the assay is ·performed. At
`this time there are -2. 0~2. 5 x l~.6 cells ·per well.
`Assays are only performed _6n cell 1 passage S24.
`. .
`At time - ·60 min, lildl!li>layers at:~ wa.~hed twice with
`. ' . . -
`.. ,~ ... ,.., .
`.
`..
`.
`2. s mL PBS, and . medium is ,·changed to 1. o mL of DMEM
`.
`.
`. •
`r
`.
`medium plus 4. 5 g/1':glucose and O'. 1%° BSA (_assay medium) .
`At O time, medium is 'as~irated and fresh assay medium,
`1.0 mL, containing test compound is added. Test compound
`is added in 50 ul volume ~ f PBS plus 0.1% BSA; controls
`are added in vehicle alone.
`Induhation is continued for
`o to 60 min.
`At termination, conditioned 'medium and monolayer
`are harvested to measure _poth exfra- and intrac~llular
`cAMP content. For extra~~flular measurement, medium is
`removed and centrifuged; td remove any cellular debris.
`For intracellular ·determlriation, after medium .removal,
`1. o mL of ice cold 95% e,ebanol is .. added to· monolayer.
`-.~~
`......
`Cells are collected by sctaping, \ysed by two cycles of
`quick freeze/thawing using liqu~~ ·N; ,'· and cell debris
`then removed by centrifugation •. -:Aliquots (l/40th well
`content) of conditiciiled inedium•.and ~thanol cell extract
`'
`.
`. .
`/t~~
`are measured in duplicate for cAMP levels using an RIA
`kit by the acetylated ptotocol.
`As above, higher patency as compared t _o glucagon is
`defined either as high~r· cAMP st~mu1ation bt ~oth the
`analog and glucagon at the same c·opcentratio:n, or the
`same cAMP st:i,mulation _by th_e analog at a ·11ower
`concentration •
`. Still other assays ·~or measurement of . enhanced
`potency to mediate insulin· release can be used.
`
`'
`
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`
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`
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`
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`
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`FRESENIUS EXHIBIT 1041
`Page 13 of 50
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`

`

`WO 91/11457
`
`PCT /US91/00500
`
`-12-
`
`5
`
`: . ~
`The abflity of the compounds to potentiate the
`release of insulin can be tested both in vitro and in
`vivo.
`Insulin released can be detected using a standard
`antibody assay both in analyzing plasma in in vivo
`studies and in analy.zing media or perfusion liquid i n
`yitro.
`For example, a useful in vitro assay uses the
`-
`pancreatic infusion assay method of Penhos, J.C., et al.
`Diabetes (1969) ll:733-738, as employed in the method of
`10 Weir, G.C., et al. J Clin Investiqat (1974) ,ai:1403-1412.
`Insulin secretion can also be measured by.the method
`described by Holst, J.J:, et al. FEBS Letters (1987)
`ll.l,.:169-174 (supra). Also _useful as an assay for
`insuli notropic effect is the measurement of stimulation
`of adenylate cyclase in the RIN 1046-38 cell line .•
`Drucker, D.J. et al., Pree Natl Acad Sci USA (1987)
`li:3434-3438 (supra).
`Inhibition of glucagon release can be shown as
`described by orstov, c., et al. Endocrinol (1988)
`rn:2009-2013; Suzuki, S., et al. Diabetes Research:
`Clinical Practice (1988) ~(Supp. 1):530 (both supra).
`Assays for Enhanced Stability to Degradation
`The therapeutic efficiency of the GLP-1 analogs of
`the invention can also be enhanced by providing analogs
`25 with increased half-lives in vivo. By "enhanced half(cid:173)
`life in vivo" is meant a demonstrated ability to resist
`degradation in the presence of plasma according to an
`assay selected from the group consisting of the
`following.
`In all assays, the plasma is prepared by
`collecting blood into heparinized tubes, placing the
`tubes on ice and centrifuging at_ about 3 ,000 rpm f or 10
`minutes in a tabletop centrifug~. The separated plasma
`is stored at 4·0 c.
`
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`FRESENIUS EXHIBIT 1041
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`

`

`W091/11457
`
`PCT /US91/00500
`
`· -13-
`
`a
`
`'
`
`•.
`
`··t
`
`A. Radiolabel s'.eguencinq:
`The GLP analog is ~p~]ied-i by radio-iodination in
`. ~. .
`..
`. . .
`position 19 using standard radi.olabeling methods. After
`exchange into RIA buffer· (50-:mM: .NaHP04 pH 7 .4,. 0 . 25% BSA
`(Armour insulin and FFA free), 0.5%., BME, 0.002%
`polylysine (Sigma 1,s,oop mw), · 0.05% .Tween 20; 0.1% NaN3
`),
`the radioiodinated pepti~e (about 105 cpm/50 mL) and cold
`uniodinated pep~ide (20 :µ1100 nM) are added into 2 ml of·
`plasma to a final concertt~ation of j nM and incubated in
`. : . .
`~'
`a circulating water bath;.-::for preset times:. Total RIA
`buffer added to plasma n~ er exceeds 5% •of total volume .
`At the end of incubatiort, . 1.0% ~acitracin (w/ v} in water
`is added to a final concentration· of 0.1% to stop the
`reaction.
`The plasma is then• .. ex.t~ac.te(i using c1s Sep-Pak to
`separate the analog and any fra~nt:s from the bulk of
`the plasma proteins. Sep-Pak cartridges (Waters} are
`washed with 2 ~L "?~ 1-pr ppanql, :f.ollowed by 2 mL of water
`and then equil.ib~a~e,d ~i~ 2 . mL of 20% CH3CN containing
`0.1% trifluoroaceti~ ,ac~.G(. (TFA) (Buffer A).
`The bacitrac;in-:tre~t~dplasma is made 20% CH3CN
`with CH3 CN contain-j,;ng o. 1·% TFA and ·is, expressed slowly
`through a 3 mt pl~stic syringe through the cartridge.
`The cartridge is then wa:shed with two 1 mL Buffer A
`25 washes and eluted with a·.,;Sing1e: 2 mL wash of 50% CH3CN
`containing o .1%, TFA (Bu.f-~er ·B) into·.,;,a siliconized 12 x 75
`glass tube. Recov~ry of the analog or fragments is more
`than 90%.
`The eluates are concentrated to 100 .. µ,l in a Speed
`vac and transferred to a .1.5 mt Epaendo:r;f tube to which a
`l mL RIA buffer rin.se of the original tube had been
`. added.
`To purify any analog or. its·. fragme~ts :when the
`analogs of GLP-1 (7-37) are used,. the concentrates are
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`FRESENIUS EXHIBIT 1041
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`

`

`W091/11457
`
`PCT/US91/00SOO
`
`· -14-
`
`5
`
`10
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`treated with 5 µl of antiserum prepared to a synthetic
`peptide corresponding to residues 24-37 which recognizes
`GLP-1, GLP-1 (7-37) but not GLP-1 (7-36). When the
`shorter forms of analogs are-used, alternate carboxy
`terminal-specific antisera (prepared in the same manner
`but using a peptide corresponding to residues 24-34, 24-
`35 or 24-36 as immunogen) are used. To this is added 100
`µl of a 10% (w/v) solution of protein A~Sepharose
`(Pharmacia) in PBS, and the mixture .is incubated
`overnight at 4°C with gentle rocking. The Sepharose is
`· then pelleted with a 5 second spin in an Eppendorf
`centrifuge at 4°C after which the pellet is washed two
`times with cold RIA buffer and four times with cold PBS .
`Polyclonal antisera were raised in New Zealand
`15 White rabbits against a synthetic peptide fragment
`corresponding to residues 24 to 37 of GLP-1 (7-37) using
`the method of Mosjoy, s. et al., J Biol Chem (1986)
`261:11880-11889.
`Initial immunizations were into the
`inguinal lymph nodes and used Freund's complete adjuvant .
`TWo subcutaneous boosts were performed. at 1 week
`· intervals after the initial immunization and used
`Freund •·s incomplete adjuvant. For a single immunization
`or boost 100 µg peptide and 100 µg methylated BSA
`dissolved in 0.3 mL phosphate-buffered saline (PBS) were
`emulsified with 0.9 mL adjuvant. · Bleeds (50 mL) began at
`week 6 after the initial immunization and continued at 1
`month intervals thereafter. Repeat boosts were performed
`as above when titers dropped noticeably from the level of
`the previous bleed.
`Serum was prepared by allowing the blood to clot
`overnight at 4°C. The clot was pelleted by
`centrifugation at 2·000 g £or 15 minutes and .the serum
`• removed. Serum is stored in aliquots at -20 or -so 0 c.
`
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`

`

`lt'f ,1""·t.:• ~,51 ~
`..
`·1··
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`.
`
`W091/11457
`
`PCT/US91/00500
`
`-15-
`
`' ;
`
`. .
`The peptides .are then eluted from the antibody
`protein-A sepharose complex with t~ee 100 µl washes of
`Buffer B. The combined 300 µl of wash are then applied
`directly to an ABI .model 477:A seqµencer used according to
`the manufacturer's instructions. · Fractions from each
`cycle are then, di ve.rted ~or counting. Counting can be
`effected in 4 mL aqueo\:ls-:•'scintillant :(ACS, Amersham) •
`The cycle at which label appe~rs indicates the
`extent of degradation from the N-terminus. If no
`degradation from the N-~erminus has occurred in the GLP-
`1 (7-3·7) analog, all of the label will appear in the 13th
`cycle, corresponding to the tyrosiri~ at position 19; if
`. ~,
`.
`.
`degradation has occurred, -~:he label will appear in
`earlier cycies.
`.,..
`B. Assay by RP~HPLC:
`·-:-~½ ·
`While the foregoing· method i~l ra cJ.ear criterion for
`exhibiting a l-0·nger -half~lite iri. ·pla~ma, a'lternative
`forms of the ass~y ,for·. t~:i,s pro11.erty can also be used.
`In one convenient assay·( the analog' can be assessed for
`degradation into f,ragmen.7s using' reverse· phase-HPLC,
`since the fragments have di-Lferent retention times from
`~'
`:
`r.i. :
`the analog per se.
`In this ~ssay, tlie analog is added to
`plasma for various ti~es and rea~v.e1ed similarly to the
`method described· above:. for. rad;o~bei: sequencing
`analysis. Specifically, the arialog ... at a concentration of
`100 nM in RIA buffer is ·spiked into 1 mL plasma to a
`final concentration of 1 nM and .aft.er incubation in 37°C
`..
`circulating water bath for vario~s preset times, the
`.
`.
`reaction is stopped by b~lnging the plasma to 0.1% (w/v)
`in bacitracin .
`.
`.The peptides are then 'purifiep :by Sep-Pak
`extraction as· describ.e.d ·.above. . Thi-:eluates are
`.
`. .
`. ·; ......
`concentrated to about 1 J11L on a S·peed-vac, diluted with 1
`mL distilled water, froze~ at 80°C and lyophilized
`
`'
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`FRESENIUS EXHIBIT 1041
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`W091/11457
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`PCT /US91/00500
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`-16- '
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`overnight. The powder is resuspended in o.s mL Buffer c
`(0.1% TFA in water) per mL starting plasma and 0.25 mL
`are injected on a Hewlett-Packard 109OL liquid
`chromatograph using an Alltech C18 column (0.45 x 25 cm;
`10 µm particle size) with a Brownlee 2 cm ClS guard
`column. The extraction is monitored at 00214 throughout
`the run and the solvent flow rate was 1 mL/minute. A
`gradient between Buffer c and Buffer D (0.1% TFA in
`acetonitrile) is set up over a 40 minute run time. The
`gradient starts at 35% .Dis held for the first 2 minutes
`after injection and then increased to 42% ·0 over 24
`minutes. The gradient is then increased to 60% Dover
`the next two minutes, held at this level for 2 minutes
`and returned to 35% Dover the next 2 minutes. The %D
`remains at 35% for the remaining 8 minutes of the run.
`Fractions are collected at 0.5 minute intervals for the
`first 30 minutes of each run and dried in a Speed-vac.
`The samples can be assaye